Using E.coli turgor pressure regulation to optimize product excretion and
prevent unwanted cytoplasmic leakage
The Challenge
The Research
Many of the most effective new treatments for cancer,
arthritis and other conditions are protein-based
biologic drugs that have to be manufactured using
living cells such as bacteria.
Dr Teuta Pilizota is a Chancellor's Fellow at the
University of Edinburgh. The research in her
laboratory focuses on developing novel tools
for quantitative observations of changes in
physiological parameters in single bacterial
cells.
Escherichia coli is a widely used host for production of
recombinant proteins for biologics. Although high
levels of protein secretion can be achieved, this is
often followed by low-yield down-stream processing
to extract functional proteins. Furthermore, at higher
protein yields (>10g/L), non-specific leakage of
cytoplasmic content is often observed, which includes
release of DNA that increases media viscosity, resulting
in further decreased yields and higher production
costs.
Downstream processing represents a bottleneck
within the Industrial Biotechnology industry and
approaches to reduce costs of this step will grow in
importance as higher product yields continue to be
achieved.
Dr Pilizota, along with FujiFilm Diosynth
Biotechnologies, applied for CBMNet Proof-ofConcept funding to investigate how turgor
pressure regulation can be used to optimize
product excretion and prevent unwanted
cytoplasmic leakage in E.coli.
The data used to secure this funding came
from a CBMNet Vacation Scholarship grant
carried out in the summer of 2014.
CBMNet Proof of Concept
Funding
The Result
The Future
Samples were obtained from several
fermentation runs performed by Fujifilm and
these confirmed that media osmolarity increases
during recombinant proteins expression.
Based on the results of the project and
collaboration, Dr Pilizota and FujiFilm have been
awarded an Industrial Biotechnology Innovation
Centre funded Scholarship for a 4 year PhD project.
A talented student, Smitha Hegde, has been
recruited to continue the work.
The project also identified and characterized the
mechanisms underpinning the export of E.coli
cell cytoplasmic contents that can be targeted to
optimise product excretion. This can be done by
adjusting the incubation period post downshock
treatment for recombinant protein extraction.
The data generated will be used to create a
protocol for controlling external media
osmolarity during production through continued
collaboration with FujiFilm.
In addition, Dr Pilizota has applied for a IB Catalyst
funding together with, Dr Meriem El Karoui , Prof
Susan Rosser and Dr Louise Horsfall. A workpackage in the IB Catalyst application contains
work based on this CBMNet project. The catalyst
work will be again supported and carried out in
collaboration with FujiFilm.
This project was funded through the Crossing Biological Membranes Network (CBMNet) by the Biotechnology and Biological
Sciences Research Council (BBSRC)
Email:
Telephone:
[email protected]
0114 222 9766
Website:
Twitter:
www.cbmnetnibb.net
@CBMNet_NIBB
“At FujiFilm we often don’t have time
to investigate the fundamental science
behind many of our processes.
Working with Dr Pilizota has allowed
us to gain some insight into the use of
novel analytical methods to gain a
better understanding of part of our
production process. This has led to a
bigger project that will lead to
improvements in our processes”
Dr Ian Hodgson, Fujifilm Diosynth
Biotechnologies
’’We have benefited from this CBMNet
award in establishing a working
relationship with an UK based IB
company that enables us to faster
translate our research into the
industrial sector. The company is
getting a new angle on establishing
control over recombinant protein
excretion, which is crucial to further
enable new innovation and ensure UK
stays one of the world leaders in IB.’’
Dr Pilizota, University of Edinburgh